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J. Biol. Chem., Vol. 280, Issue 4, 3051-3059, January 28, 2005

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Structures of dCTP Deaminase from Escherichia coli with Bound Substrate and Product

REACTION MECHANISM AND DETERMINANTS OF MONO- AND BIFUNCTIONALITY FOR A FAMILY OF ENZYMES^*

Eva Johansson¶, Mathias Fanø||¶, Julie H. Bynck, Jan Neuhard**, Sine Larsen, Bent W. Sigurskjold||, Ulla Christensen, and Martin Willemoës

From the Centre for Crystallographic Studies, Department of Chemistry, University of Copenhagen Universitetsparken 5, DK-2100, Copenhagen, Denmark, the European Synchrotron Radiation Facility, BP 220, F-38043 Grenoble Cedex, France, the ^||Department of Biochemistry, August Krogh Institute, University of Copenhagen Universitetsparken 13, DK-2100, Copenhagen, Denmark, the ^**Department of Biological Chemistry, Institute of Molecular Biology, University of Copenhagen Sølvgade 83H, DK-1307, Copenhagen, Denmark, and the Department of Chemistry, Laboratory for Physical Chemistry, University of Copenhagen Universitetsparken 5, DK-2100, Copenhagen, Denmark

dCTP deaminase (EC 3.5.4.13) catalyzes the deamination of dCTP forming dUTP that via dUTPase is the main pathway providing substrate for thymidylate synthase in Escherichia coli and Salmonella typhimurium. dCTP deaminase is unique among nucleoside and nucleotide deaminases as it functions without aid from a catalytic metal ion that facilitates preparation of a water molecule for nucleophilic attack on the substrate. Two active site amino acid residues, Arg¹¹⁵ and Glu¹³⁸, were identified by mutational analysis as important for activity in E. coli dCTP deaminase. None of the mutant enzymes R115A, E138A, or E138Q had any detectable activity but circular dichroism spectra for all mutant enzymes were similar to wild type suggesting that the overall structure was not changed. The crystal structures of wild-type E. coli dCTP deaminase and the E138A mutant enzyme have been determined in complex with dUTP and Mg²⁺, and the mutant enzyme also with the substrate dCTP and Mg²⁺. The enzyme is a third member of the family of the structurally related trimeric dUTPases and the bifunctional dCTP deaminase-dUTPase from Methanocaldococcus jannaschii. However, the C-terminal fold is completely different from dUTPases resulting in an active site built from residues from two of the trimer subunits, and not from three subunits as in dUTPases. The nucleotides are well defined as well as Mg²⁺ that is tridentately coordinated to the nucleotide phosphate chains. We suggest a catalytic mechanism for the dCTP deaminase and identify structural differences to dUTPases that prevent hydrolysis of the dCTP triphosphate.

Received for publication, August 19, 2004 , and in revised form, November 10, 2004.

The atomic coordinates and structure factors (codes 1XS4, 1XS1, and 1XS6) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

^* This work was funded by the Danish National Research Foundation and the Danish National Science Research Council. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^¶ These authors contributed equally to this work.

To whom correspondence should be addressed: Centre for Crystallographic Studies, Dept. of Chemistry, University of Copenhagen, Universitetsparken 5, DK-2100, Copenhagen, Denmark. Tel.: 45-35-32-02-39; Fax: 45-35-32-02-99; E-mail: martin{at}ccs.ki.ku.dk.

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